This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Inhibition of the interaction between Interleukin-2 (IL-2), an immunoregulatory cytokine, and its receptor alpha chain (IL-2R alpha) using small molecules is an extensively studied problem, and there exist two examples of small molecular inhibitors of IL-2, both of which have been characterized by X-ray crystallography. Both inhibitors bind at the same site on IL-2, which coincides with the binding interface to IL-2R alpha and involves interactions with some of the amino acid residues on IL-2 known to comprise the energetic hot spot for IL-2/IL-2R alpha binding. It was shown that the small molecule binding site can be divided into two subsites, the first being a largely polar and rigid pocket, the second a highly adaptive hydrophobic region. The binding energies that different portions of the known inhibitors derive from their interactions with the protein have not been systematically elucidated. We will measure experimental binding energies and binding orientations for different molecular fragments derived from these known IL-2 inhibitors, using quantitative biochemical and biophysical assays as well as X-ray crystallography, and will compare the results with those obtained computationally using the same fragments as probes. The results will provide new information on the physicochemical and structural features that render a difficult PPI site druggable, which we will use to further refine our computational method.